Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 Integrins and Expression of Ligand-Induced Binding Site

Abstract

AbstractEchistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.